Institute of Translational Immunology and Research Center for Immune Therapy,

University Medical Center Mainz, Germany

Division of Gastroenterology, Beth Israel Deaconess Medical Center

Harvard Medical School, Boston, USA

Detlef Schuppan

Liver Forum, EASL, Amsterdam, April 18, 2017

Liver fibrosis in NASH: A Roadmap for Drug Discovery and Pharmacotherapy

Research

Center for

Immune Therapy

HARVARD

MEDICAL

SCHOOL Institute for Translational Immunology

I have no conflicct of interest to declare

Noncirrhotic fibrosis

Liver related death

Relevance of advanced liver fibrosis/cirrhosis as endpoint

Cirrhosis Decompensation

HCC

HCC

Induction of reversal

TIMP-1

TIMP-2

MMP-1/3/13 matrix accumulation

fibrotic liver collagen- synthesis

activated myofibroblast

HSC

endothelium

repetitive damage

(second hit)

progenitor cell, cholangiocyte, hepatocyte

Toxins Toxic bile salts (Auto-) Immunity HBV, HCV Ox. stress, ROS Insulin resistance Microbiome, nutrients

Cirrhosis organ failure

genetic predisposition

Cirrhosis and HCC

common pathways & immune environment !

Fibrogenesis is a Multicellular Process

normal liver

quiescent stellate cell

portal or perivascular fibroblast

Schuppan and Afdhal, Lancet 2008

Schuppan and Kim, JCI 2013

MF

T

macrophage

Reversibility of advanced fibrosis after removal/suppression of the

primary causal hit

Baseline: no cirrhosis

Better 105/252

Worse 12/252

Baseline: cirrhosis

Better 71/96

(≥2 stages: 70/71)

Marcellin P et al, Lancet 2013

Cirrhosis Regression with Longterm Tenofovir Treatment 1-5 yr extension of 48 week tenofovir trial (Marcellin P et al, NEJM 2008)

489/615 pts (76%) included 5 yr biopsy: 348/489 (71%)

Antifibrotic approaches

B

IL-17, IL-22, IFN

Endothelial cell

T cell

Macrophage (monocyte)

Cholangiocyte

Hepatocyte

Damaged hepatocyte

Hepatic stellate cell

Myofibroblast

A

Schuppan and Kim, J Clin Invest 2013

Mehal and Schuppan, Sem Liver Dis 2015

• complex interactions between cells and cytokines, chemokines, extracellular matrix

• anti- or profibrotic effects are context-dependent

• unambiguous targets are rare

Role of activated cholangiocytes in fibrosis progression

activated progenitor cells/ cholangiocytes

activated myofibroblasts

PDGF-BB, ET-1 TGFb1, TGFb2 CTGF, MCP-1

HGF, bFGF, IL-6, Shh

Progenitors (“activated cholangiocytes”) as driving force of fibrogenesis

Proliferation stimulus

inflammation

Hepatocyte growth arrest / lipoapoptosis

Rise of fibrogenic progenitor cells ductular reaction EMT-like changes

2nd hit

e.g. oxidative stress in NAFL

General mechanism in fibrosis ≥ F2 FCH: Davies SE et al, Hepatology 1991

ALH: Ray MB et al, Liver 1993

Various CLD: Lowes KN, Am J Pathol 1999

HCV: Clouston A et al, Hepatology 2005

NASH: Richardson MM et al, Gastroenterology 2007

Integrin αvβ6

αvβ6 inhibitors Wang et al, Hepatology 2007; Popov Y et al, J Hepatology 2008; Patsenker E et al, Gastroenterology 2008 Cirrhosis/HCC

Tweak

Kuramitsu K et al, Hepatology 2013 Fn14

Monocyte

MC

MΦ2

MΦ1

classically activated M1 macrophage

alternatively activated M2 macrophage

Phagocytosis of debris and apoptotic cells

Generation of pro-inflammatory cytokines

“Resolution of fibrosis” Immune-activating & cancer-suppressive

Promotion of angiogenesis

Generation of anti-inflammatory cytokines

“Promotion of fibrosis” Immune-suppressive & cancer promoting

Pharmacological repolarization of macrophages

Targeted modulation

Macrophages of „wounds that do not heal“ = fibrosis- and tumor-associated macrophages

Duffield JS et al, JCI 2005 Fallowfield JA, J Immunol 2007 Duffield JS et al, JCI 2007 Popov Y et al, Am J Physiol 2010 Ramachandran S et al, PNAS 2012 Schuppan and Kim, JCI 2013 Mehal and Schuppan, Sem Liver Dis 2015 Ramachandran P et al, Sem Liver Dis 2015

Pro-resolution macrophage

CD11b+ F4/80+ Ly6Clow

Mechanism based antifibrotic therapies in clinical development

Drugs in phase I-II clinical trials to address fibrosis (1)

ECM, EMT, Progenitor activation (inflammation, hepatocyte apoptosis)

•Gilead: GS6624 (Simtuzumab): α-Loxl2 Mab (>700 patients with

stage 2 NASH or PSC)

• Gilead: GS9654 (Selonisertib): Ask1 (apoptosis signaling kinase 1)

inhibitor (70 patients with stage 2 NASH)

• Biogen-Stromedix: STX-100: α-Integrin αVβ6 Mab

• Biogen-Idec: anti-Tweak

• Sanofi-Genzyme: Fresolimumab: α-TGFβ Mab

• Pfizer-Fibrogen: FG3019: α-CTGF Mab

• Novartis: Seculizumab, α-IL-17 Mab

• Conatus: Emricasan: Caspase inhibitor (>250 pts with NASH)

• Boehringer-Ingelheim: VAP-1 antagonist (>200 pts with NASH)

Schuppan and Kim, J Clin Invest 2013; Mehal and Schuppan, Sem Liver Dis 2015 Torok et al, Hepatology 2015 Trautwein et al, J Hepatol 2015

Drugs in phase I-II clinical trials to address fibrosis (2)

(M2) Macrophages, hepatic stellate cells

• Janssen: Carlumab: α-MCP-1/CCL2 Mab

• Pfizer: Selzentry: CCR5 antagonist

• Tobira/Allergan: Cenicriviroc: CCR2/CCR5 antagonist (289

patients with NASH)

• BMS: Peg-FGF21

• Novartis: QUAX576: α-IL-13 Mab

• Sanofi: SAR156597: α-IL-4/IL-13 Mab

• Isis and own group: IL4Rα1, IL13Rα1……. antisense-DNA

• Promedior: RM-151: rec. Pentraxin-2 (SAP)

• Novo Nordisc: GLP-1 agonist/Semaglutide) (300 pts with

NASH)

Schuppan and Kim, J Clin Invest 2013; Mehal and Schuppan, Sem Liver Dis 2015 Torok N et al, Hepatology 2015 Trautwein et al, J Hepatol 2015

An anti-inflammatory agents are not necessarily antifibrotic, examples:

• anti-TGFb1: blocks fibrosis, enhances inflammation

• anti-CCR2, CCR5… : blocks inflammatory and restorative macrophage infiltration/activation, HSC activation

Current and evolving options for fibrosis assessment

Elastography – crude

assessment, not dynamic

Liver Biopsy – crude

assessment, not dynamic

Fibrosis serum markers

Imaging

→ validated markers of

fibrogenesis and fibrolysis

→ targeted and quantitative

imaging of fibrogenesis

Sampling variability in staging & grading

Regev et al., Am. J. Gastroenterol. 97, 2614, 2002

HCV, laparoscopic biopsy of right and left liver, n=124, Metavir-score

Difference n %

1 stage 41/124 33.1

2 stages 3/124 2.4

1 grade 30/124 24.2

2 grade 2/124 1.6

cirrhosis vs. stage 3 18/124 14.5

liver biopsy

samples only

1/ 50.000 of

the whole liver

1 stage discordance

NASH > 40% Ratziu V, Gastroenterology 2005; Merriman RB et al, Hepatology 2006

PBC/PSC > 60% Olsson R et al & Garrido MC et al, J Clin Pathol 1995 & 1996

Comparison of 6 biomarker scores in 180 pts with CHC (F0-1 vs. F2-4)

MP3: PIIINP, MMP-1

APRI: AST / platelets

Forns: GT, cholesterol,

platelets, age

Fibrotest: age, Bili, GT,

-globulin, haptoglobin,

a2M

Hepascore: age, gender,

a2M, HA, Bili

Fibrometer: age, INR,

platelets, AST, urea,

a2M, HA

Leroy V et al, J Hepatol 2007

All with AUROCs 0.80-0.85 = only modest tests to distinguish no/mild vs. significant fibrosis

fibrolysis

MMPs….

degradation

Rosenberg W et al, Gastroenterology 2004; Parkes J et al, Gut 2010;

Leeming D et al, Alim Pharm Ther 2013; Karsdal M et al, Am J Physiol 2015

fibrogenesis: P3NP, ProN5, TIMP-1, hyaluronic acid

Direct markers of fibrosis dynamics

precursor synthesis

Biological plausibility:

matrix degradation/turnover: C3M, C4M,

C5M, C6M, lumican, laminins, tenascin

Best current marker of fibrogenesis: ProC3

Marker(s) of fibrolysis needed

But: degradation fragments also derive from

freshly synthesized matrix proteins ELF-Panel

NB biomarkers - established assays –validation for liver:

C5M (MMP-mediated type V collagen degradation)

C6M (MMP-mediated type VI collagen degradation)

ProC3 (type III collagen formation) - fibrogenesis

ProN4 (type IV collagen formation)

ProC5 (type V collagen formation)

ProC6 (type VI collagen formation, adipokine) –

fibrogenesis, adipose tissue fibrosis (NASH)

UMCM biomarkers of progression or reversal - derived from serum iTRAQ and Somascan proteomics:

WDR85, WDR90, Ephrin B3, A9*, IB3*, PR8*, MK3* - fibrogenesis

IB3*, PR8*, MK3*, A2*, A14*, CS17*, CS26*, TP2* - fibrolysis

* in validation, listed for patent protection

Institute for Translational Immunology

Nielsen M et al, Liver Int 2015 Karsdal MA et al, Am J Physiol 2016 Leeming D et al, submitted Surabattula R et al, submitted

Procollagen III

Intact

Degra- dation

C3M

Formation

PIIINP

Pro-C3

Pro-C3-Ab

PIIINP-Ab C3M-Ab

each variant of the same protein carries unique information

Procollagen type III processing

Nonresponders to HCV-antiviral therapy

Ishak fibrosis score 2-4

Treatment duration: 52 weeks with

follow up biopsies (209/265=79%)

Placebo controls n=89

Farglitazar 0.5 mg/d: n=88

Farglitazar 1.0 mg/d: n=88

Histological quantification:

α-SMA, collagen (SR morphometry)

Non-TZDD PPAR- agonist with

100-1000fold higher activity than

Pio-/Tro-/Rosi-glitazone

no overall effect on fibrosis !

collagen α-SMA

Placebo controls +49% 27%

Farglitazar 0.5 mg/d: +58% 27%

Farglitazar 1.0 mg/d: +52% 31%

pre post

• > 20.2 ng/ml: selection criterion for responders

• a decline in serum levels indicates antifibrotic effect

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Karsdal MA et al, Am J Physiol 2016

Pro-C3 identifies subjects who responded to antifibrotic therapy

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Follow up on Pro-C3 levels

A9 A2

Markers of fibrogenesis and fibrolysis:

A2, A9 and A14: cell membrane molecules involved in ECM remodeling

NS, healthy ctr

LTX: post

transplant with

progression to

cirrhosis within 5 yr

Fibrolysis Fibrogenesis

A9 A2 BAV: before

antiviral Tx for HCV

AAV: 24 w after

highly effective

antiviral Tx for HCV

Surabattula R et al, unpublished

• (early) Cirrhosis is reversible when the major fibrogenic (inflammatory) trigger is eliminated (HepB, HepC, ai-Hep)

• This may even be possible for (decompensated) cirrhosis

• Most NASH drugs target the hepatocyte and its metabolic derangement, possibly with secondary antifibrotic effects

• Some drugs target inflammation, but this does not necessarily correlate with antifibrotic activity

• Other drugs address multiple cells and net effects are difficult to predict

Summary (1)

• Major antifibrotic targets are related to fibrogenic cholangiocytes, macrophages and hepatic stellate cells

• Several (pharmacological) therapies that may inhibit progression and speed up reversal have entered the clinic

• Biologically plausible markers of fibrosis, fibrogenesis and fibrolysis to stratify patients and noninvasively monitor treatment response are being developed

• This should permit short and slim POC studies, testing of combinations and a personalized antifibrotic therapy

Summary (2)